GSM6685107: CUT&RUN, p53,Trp53_KO,Rep2; Mus musculus; OTHER
CUT&RUN protocol and all buffer recipes used were adapted from Skene et. al. (Skene et al., 2018) and performed in 0.2ml tubes. Digitonin concentrations were selected based upon trypan blue permeabilization assessment conducted one day prior to experiment as previously described (Skene et al., 2018). Briefly, 200,000 cells were washed and immobilized on 10uL of activated Concanavalin A-coated magnetic beads per condition. Captured cells were resuspended in 0.2ml of antibody buffer containing primary antibody diluted at 1:100. Tubes were rotated 2 hours at 4°C to promote antibody binding to target protein. Cells were then washed with 0.2ml of digitonin wash buffer and resuspended in 0.2ml of digitonin wash buffer containing pA-MNase (expressed and purified in house) diluted to a final concentration of ~0.8ng/uL. Tubes were rotated 1 hour at 4°C to promote protein A binding to primary antibody. Buffer was discarded and replaced with 150uL of digitonin wash buffer. Tubes were cooled to 0°C for 5 minutes, then supplemented with CaCl2 to promote MNase digestion. After 30 minutes, 150uL of 2X STOP buffer containing chelating agents and heterologous yeast spike-in DNA (kind gift Dr. Steven Henikoff, Fred Hutchinson Cancer Research Center) was added to stop the reaction. Cells were incubated at 37°C for 10 minutes to promote release of soluble chromatin fragments, then centrifuged at 16,000xg for 5 minutes at 4°C to pellet cells. Supernatant containing soluble chromatin fragments was transferred to new tubes for DNA extraction. Phenol chloroform extraction was conducted on fragments bound by transcription factors to capture small fragments that may not be captured by column-based extraction methods. Column-based extraction (QIAgen minElute) was conducted for histone CUT&RUN experiments since minimum fragment sizes from histone CUT&RUN experiments typically exceed minimum ranges for column-based DNA extraction. DNA libraries were prepared by PMGC using either NEBNext Ultra II DNA library kit (New England Biolabs) or ThruPLEX DNA-seq kit (Takara Bio USA) as per manufacturer's instructions. Unique molecular indexes were included with p53 CUT&RUN experiments. Libraries were sequenced to obtain >40M paired-end reads / sample on an Illumina NovaSeq6000.